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Hypoxia suppressed copper toxicity during early development in zebrafish embryos in a process mediated by the activation of the HIF signalling pathway Jennifer A Fitzgerald, Hannah M Jameson, Victoria H Dewar Fowler, Georgia L Bond, Lisa K Bickley, Tamsyn M Uren Webster, Nicolas R. Bury, Robert J Wilson, and Eduarda M. Santos Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b01472 • Publication Date (Web): 28 Mar 2016 Downloaded from http://pubs.acs.org on March 30, 2016
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Hypoxia suppressed copper toxicity during early
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development in zebrafish embryos in a process
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mediated by the activation of the HIF signalling
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pathway
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Jennifer A. Fitzgerald1 2*, Hannah M. Jameson1, Victoria H. Dewar Fowler1, Georgia L. Bond1,
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Lisa K. Bickley1, Tamsyn M. Uren Webster1, Nic R. Bury3, Robert J. Wilson1, Eduarda M.
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Santos1*.
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Biosciences, College of Life & Environmental Sciences, Geoffrey Pope Building, University of
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Exeter, Exeter, EX4 4QD, UK 2
Centre for Environment, Fisheries and Aquaculture Science, Barrack Road, The Nothe,
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Weymouth, Dorset, DT4 8UB, UK 3
83 Franklin-Wilkins Building, King’s College London, London, SE1 9NH
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*Corresponding authors
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ABSTRACT: Hypoxia is a global and increasingly important stressor in aquatic ecosystems,
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with major impacts on biodiversity worldwide. Hypoxic waters are often contaminated with a
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wide range of chemicals but little is known about the interactions between these stressors. We
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investigated the effects of hypoxia on the responses of zebrafish (Danio rerio) embryos to
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copper, a widespread aquatic contaminant. We showed that during continuous exposures copper
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toxicity was reduced by over 2-fold under hypoxia compared to normoxia. When exposures were
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conducted during 24h windows, hypoxia reduced copper toxicity during early development and
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increased its toxicity in hatched larvae. In order to investigate the role of the hypoxia signalling
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pathway on the suppression of copper toxicity during early development, we stabilised the
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hypoxia inducible factor (HIF) pathway under normoxia using a prolyl-4-hydroxylase inhibitor,
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dimethyloxalylglycine (DMOG) and demonstrated that HIF activation results in a strong
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reduction in copper toxicity. We also established that the reduction in copper toxicity during
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early development was independent of copper uptake, while after hatching, copper uptake was
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increased under hypoxia, corresponding to an increase in copper toxicity. These findings change
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our understanding of the current and future impacts of world-wide oxygen depletion on fish
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communities challenged by anthropogenic toxicants.
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INTRODUCTION
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Hypoxia is one of the most significant stressors affecting aquatic systems worldwide, and its
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severity and prevalence are projected to rise due to increases in nutrient input and climate change
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[1]. With rapid industrialisation and population growth, agricultural, industrial and domestic
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effluents containing a wide range of potentially toxic chemicals and nutrients are also
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increasingly being discharged into aquatic systems, with long term consequences for aquatic
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organisms [2]. Therefore, environmental pollutants and hypoxia often co-occur in aquatic
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systems and, consequently, their potential interacting effects on wildlife must be considered.
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To date, few studies have investigated whether chemical toxicity to fish is modified by the
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availability of oxygen in the water. The chemicals considered in existing studies include
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polyaromatic hydrocarbons [3], polychlorinated biphenyls [3-9], phenols [10,11], ammonia
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[10,12], oestrogenic chemicals [13] and toxic metals [10,14-18], and evidence suggests that
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alterations in chemical toxicity are highly likely to occur. However, data are often contradictory
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and hypoxia-induced changes in chemical toxicity appear to vary widely as a function of the
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chemicals being considered, the model species and its life stage, highlighting this as an essential
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area for further research.
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Among aquatic contaminants, metals are particularly widespread and reach highly toxic
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concentrations in areas associated with mining and industrial activities [19]. Recent analysis of
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the relative threat posed by metals to aquatic organisms has identified copper as the most
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significant metal pollutant in UK waters [20]. Existing studies focusing on the toxicological
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effects of metals in combination with hypoxia have included copper [2], cadmium [15,16], zinc
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[10,21], nickel [17] and lead [10], and have found a suppression of the natural response to
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hypoxia in the presence of metals, or increased metal toxicity. For copper (Cu), limited data is
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available but, generally, an increase in toxicity has been suggested. For example, for carp, copper
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toxicity was shown to increase when exposures occurred under hypoxia [2, 22], and similarly, in
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the mayfly, Ephoron virgo, copper-induced mortality increased under hypoxia [23]. However,
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these studies have not provided any insight on the mechanisms responsible for hypoxia-induced
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alterations in the observed toxicity.
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Here, we present the first study, to our knowledge, investigating the influence of combined
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exposure to hypoxia and copper on embryonic development, using the zebrafish (Danio rerio) as
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a model fish species. Embryos are particularly vulnerable to chemical exposures due to the
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sensitive nature of the developmental processes during embryogenesis. In addition, fish embryos
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are more likely to be exposed to hypoxic conditions than other life stages: eggs of many fish
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species are deposited in areas of slow water flow and/or high nutrient input, where the co-
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occurrence of environmental contaminants and hypoxia are likely. Furthermore, embryos lack
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the ability to avoid unfavourable conditions by moving away from contaminated areas, and rely
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principally on biochemical response pathways to survive periods of hypoxia. This study aimed to
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determine the effects of hypoxia on copper toxicity throughout this vulnerable life stage, and the
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relative susceptibility of developing embryos at various stages of development to these combined
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stressors. Further, the mechanisms responsible for the effects of hypoxia on copper toxicity were
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investigated in order to generate a mechanistic understanding of the interactions between copper
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toxicity and hypoxia, helping to support predictive toxicology in the future.
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MATERIAL AND METHODS
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Copper exposures under normoxia and hypoxia
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Eggs were collected from a breeding population of zebrafish (wild-type WIK strain) according
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to the procedures described in SI. Fertilised embryos (20 embryos per tank, triplicate tanks per
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copper concentration) were exposed to concentrations of copper ranging from 0-0.1 mg Cu/L
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from 4-100hpf, to generate cumulative mortality curves under normoxic and hypoxic conditions.
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For exposures conducted under normoxia (98.4% ±0.12 air saturation), water was aerated for 1
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hour before the start of the exposures, and allowed to equilibrate to 28°C. For exposures
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conducted under hypoxia (45.3% ±0.21 air saturation), water was aerated with nitrogen for 1
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hour, to remove dissolved oxygen, allowed to equilibrate to 28°C and then mixed with aerated
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water at the appropriate proportion to obtain the desirable level of air saturation. All tanks were
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filled with 600ml of water containing the appropriate air saturation and copper concentration. A
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large volume of water (30ml of water per embryo) was used to avoid changes in the water
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characteristics caused by the metabolic activity of the embryos. For hypoxia treatments, tanks
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were sealed with a glass plate to prevent gas exchange and re-oxygenation of the water. After
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each 24h exposure period, the percentage of air saturation was immediately measured in each
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exposure tank using a calibrated oxygen meter, according to the manufacturer’s instruction
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(Stathkelvin Instruments Oxygen Meter, model 781, UK). Mortalities and hatching (for the 76
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and 100hpf observations) were recorded for each tank. After observations were completed at the
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end of each 24h period, water was completely replaced with freshly made exposure water at the
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appropriate air saturation and copper concentration, as described above.
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In order to investigate the susceptibility of the various stages of embryo development to
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combinations of copper and hypoxia, exposures were conducted during specific developmental
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windows at 24 hour intervals (4-28, 28-52, 52-76 and 76-100hpf) to form mortality curves, using
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a range of copper concentrations (from 0.01-0.4 mg Cu/L), under hypoxia and normoxia. These
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concentrations include environmentally relevant concentrations common in contaminated
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environments. Embryos (20 per tank) were incubated under control conditions (98.3% ±0.16 air
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saturation, 0 mg Cu/L) up to the start of the exposure period, and terminated immediately after
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the experiments. The percentage of mortalities was recorded after each 24h exposure experiment,
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and the percentage of hatched embryos was recorded for the 52-76hpf exposure window. All
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experiments were conducted in triplicate, with the exception of the exposures conducted during
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the developmental period of 76-100hpf period, which were carried out in quadruplicate.
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Effects of the biochemical activation of the HIF pathway on copper toxicity during early development We exposed embryos to copper in the presence of a prolyl-4-hydroxylase inhibitor,
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dimethyloxalylglycine (DMOG), which suppresses oxygen-induced HIF degradation, therefore
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activating the HIF signalling pathway independently of the presence of oxygen [24]. Embryos
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were exposed to either 0 or 0.07 mg Cu/L in normoxic water or in water containing 20µM
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DMOG (D3695 SIGMA, UK). In parallel, embryos were also exposed to hypoxia alone, and to
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hypoxia in combination with 0.07 mg Cu/L. Each exposure tank contained 100ml of exposure
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water and 10 embryos, and 6 independent tank replicates were included for each treatment group.
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The concentration of DMOG used was chosen based on a preliminary experiment where a range
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of concentrations (0.2 to 200µM) were tested in comparison with a range of hypoxia treatments.
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The concentration selected was the highest concentration of DMOG where no developmental
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effects were observed, resembling that of the level of hypoxia used in this experiment (49.6%
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±0.51 air saturation) which also does not cause any measurable developmental effects in exposed
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embryos.
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Copper uptake and quantification of gene expression
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We hypothesised that hypoxia may cause changes in copper uptake, resulting in differential
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toxicity. To investigate this, embryos were exposed to 0 or 0.024 mg Cu/L (this concentration
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caused approximately 10% mortality in the continuous copper exposure) for 24h under hypoxic
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or normoxic conditions, for the 4-28, 28-52, 52-76, 76-100hpf developmental windows, as
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described above. Copper concentrations in exposed embryos and in the water were measured by
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ICP-MS. A full description of the experimental setup, sample collection and copper
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measurements is provided in SI.
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Real-time quantitative PCR (RT-QPCR) was used to quantify the transcript profiles of exposed
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embryos for target genes known to be involved in the responses to copper and/or hypoxia in fish.
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These included genes involved in pH regulation and gas transport (carbonic anhydrase II (ca2),
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carbonic anhydrase IX (ca9)), copper uptake, transport and/or storage (cytochrome c oxidase
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copper chaperone (cox17), ATPase Cu++ transporting, alpha polypeptide (atp7a), metallothionein
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2 (mt2)) and oxidative stress (catalase (cat), superoxidase dismutase 1 (sod1), glutathione-s-
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transferase pi 1 (gstp1), glutathione S-transferease alpha like (gstaI) and glutathione peroxidase 1
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a (gpx1a)). Ribosomal protein l8 (rpl8) was used as a control gene for normalisation purposes.
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This gene has been shown to remain stable across tissue types and experimental conditions [25],
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including under hypoxia in cyprinids [26] and during embryogenesis in zebrafish, in the presence
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or absence of exposure to silver [27]. Quantitative RT-QPCR assays for each target gene were
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optimised as previously described [28] and detailed information for each assay is provided in
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Table S1. A detailed description of these methods is given in SI.
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Statistical analysis
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Statistical analysis to test for differences between the proportion of mortality and hatching
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following exposure to copper under either hypoxia or normoxia were conducted using
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generalized linear models in R [29]. A separate model was carried out for each time period after
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fertilisation, using a quasibinomial error structure and logit link to test for effects of copper
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concentration on the proportion of mortality (as a continuous variable), hypoxia or normoxia (as
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a categorical variable) and the interaction between the two. Minimum adequate models were
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derived by model simplification using F tests based on analysis of deviance [30]. A similar
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approach of model simplification of generalized linear models with quasibinomial error structure
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was used to test for the effects on the proportion of hatching of copper, hypoxia or normoxia and
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their interaction. F tests reported refer to the significance of removing terms from the models.
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For the data investigating the effects of DMOG, a Kruskal-Wallis test was used to test for
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overall treatment effects, followed by pairwise Wilcoxon tests correcting for multiple
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comparisons using the Holm method. Gene expression data was first scrutinised by Chauvenet’s
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criterion to detect outliers for each gene and these were subsequently removed [31]. For both
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transcript profiles and quantification of copper in exposed embryos, data that did not meet the
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normality and equal variance criteria was log transformed before a one-way analysis of variance
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was performed. When a significant effect was identified, pairwise comparisons to determine
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which groups differed were conducted using the Holm-Sidak post hoc test. All data was
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considered statistically significant when p
